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标题: 冰冻切片的注意事项 [打印本页]

作者: dingwei    时间: 2001-5-30 12:48     标题: 冰冻切片的注意事项

1. 速冻:是冰冻切片的关键,因为只有速冻才能减少组织内的冰晶,最好的办法是将冷冻头放入液氮内冷冻。在液氮里冷冻要注意不要冻过头,只要看到3/4的组织已经冻住就可以了,待冷冻头拿出后刚好全部冻住,否则就会引起组织发脆,或冷冻头与组织分离。
2. 固定:切片后应立即放入甲醇中固定,不能等切片干后再固定,这样容易造成细胞退变。固定液有很多,经我们实践后发现甲醇作用快,收缩小,染色较清晰,是冰冻切片最理想的固定液。
3. 包埋剂:可用普通胶水代替,但要加入适量的水。太稀了,容易损伤切片刀;太稠了,粘在切片上不容易洗掉,影响切片的质量。
4. 冰冻用切片刀一定要磨快,并且要磨直,否则切出的片子就不平整。
5. 组织发脆后可等几分钟后再切,也可用手在组织上摸几下,如果用手摸,最好戴手套,或用玻片间的纸夹在手与组织之间,以防感染。
冷冻室的温度冬天一般设在19℃左右,夏天设在22℃左右。


作者: njmuzyl    时间: 2002-5-1 12:46     标题: 冰冻切片的注意事项

万事开头难!我正准备做皮肤病理冰冻切片,现有一个问题要请教坛主
在取过组织后,如何速冻,需要把组织浸入液氮中吗,多长时间,能不能
长期放? :em06:
作者: dingwei    时间: 2002-5-12 12:59     标题: 冰冻切片的注意事项

能,如果是马上做冰冻切片,只要将组织浸入液氮中,不时拿出看一下(5-15秒),只要3/4冻住了就可以了,不然会过头。不过过头关系也不大,只要复温就可以了。
作者: 紫晶艾玛    时间: 2002-7-21 14:03     标题: 冰冻切片的注意事项

我用的是莱卡的冰冻切片机,也试着用普通胶水代替过包埋液,发现效果还是不错的,倒是加水后很难切了,也许老大说得对,我加多的:(
作者: shanghainese    时间: 2004-9-29 00:43     标题: 冰冻切片的注意事项

引用:
下面引用由江平文雨2004/09/17 04:34pm 发表的内容:
我想请较一下“速冻才能减少组织内的冰晶”原理是什么?因为细胞的冻存是采用慢冻的方法才能减少冰晶的产生,为什么组织刚好相反呢?
书上有。
作者: ylzhao    时间: 2004-10-16 00:02     标题: 冰冻切片的注意事项

[这个贴子最后由ylzhao在 2004/10/16 09:11am 编辑]

关于冷冻理论原文剪贴
原文作者:Mark Donovan and Henry Preston
Theory of freezing
The reasons for freezing a tissue sample are to provide a hardened matrix for sectioning or to preserve the morphological, biochemical or immunological properties of a cell or tissue. The use of a low temperature can eliminate many of the problems associated with standard practices of chemical fixation and paraffin or resin embedding. In practice however, the process of transforming water into ice can dramatically alter the physical and chemical structure of cells and tissue.
When pure water is cooled, large hexagonal ice crystals form as a result of homogeneous and heterogeneous nucleation and subsequent growth of ice crystal nuclei. The transformation of water into ice crystals occurs at a temperature of O°C and a pressure of 1 atmosphere. Where a very high rate of cooling exists, cubic rather than hexagonal ice crystals will form. These are far smaller and produce less distortion on formation. If the rate of cooling is increased further to within the order of 104K/sec small volumes of water can be solidified without the formation of ice crystals at all.
1 This solid form of ice, known as vitreous ice, exists in a temperature dependent, irreversible phase transition with cubic ice, hexagonal ice and water.
2 The critical temperatures at which these phase transitions occur have only been accurately determined for pure water3 but the values for intracellular water are considered to be significantly higher (fig 1).4-5
(fig 1).4-5:http://home.primus.com.au/royellis/fig1.html
The formation of large, hexagonal ice crystals as the result of slow freezing occurs firstly in the less concentrated extracellular fluid. This produces an osmotic difference between the extracellular and intracellular fluid which results in a loss of intracellular water and the subsequent shrinkage of the cell as the osmotic balance returns. The increased ionic concentration within the cell ruptures membranes and denatures the protoplasm. The eventual formation of ice crystals within the cell, when residual intracellular fluid freezes, may then mechanically fracture the cell. These effects are collectively known as ice crystal artefacts.
The damage produced by ice crystals depends upon the size and type of crystal formed during the freezing process. Large hexagonal ice crystals will produce major structural damage to cells and tissue whilst smaller cubic ice crystals cause less cellular damage. Ideally an extremely rapid cooling rate should be used as this will produce vitreous ice without crystal damage. The aim when freezing the sample, therefore, is to limit ice crystal formation as much as possible through control of the cooling rate.
Factors which effect the cooling rate of the sample include:

the absolute temperature of the cryogen.
the heat capacity of the cryogen.
extent of contact between cryogen and sample.
degree of heat exchange between cryogen and sample.
diffusion of heat through the sample.
Freezing of the specimen will require selection of an appropriate cryogen and procedures which maximise the heat exchange between cryogen and specimen. The cooling rate attainable is particularly influenced by specimen size. Above a critical specimen size optimal freezing will only occur to a certain depth and the cooling rate in the deeper parts of the sample will be slow enough to allow the formation of hexagonal ice crystals with subsequent tissue damage. It is possible to reduce the level of ice crystal formation through the use of cryoprotectants which reduce the rate of ice crystal nucleation through freezing point depression.






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